Method for the preparative-scale production of fatty acids from biomass by in-situ extraction, reaction and chromatography using compressed gases
The invention relates to a method for the preparative-scale production of fatty estersxe2x80x94for producing fatty acids, preferably polyunsaturated fatty acids (PUFAS)xe2x80x94from biological sources by continuous in-situ extraction, reaction and chromatography using compressed gases.
In nature, PUFAs, in addition to oleic acid, occur in relatively high concentrations in linseed oil, hazelnut oil, poppy seed oil, hemp seed oil and fish oils. Numerous attempts have already been made to produce these valuable substances from biological sources of these types and to isolate them with a greater or lesser degree of purity. However, since the PUFAs are generally chemically bound as esters in lipids, in addition to extraction, conversion to the free acids (hydrolysis) or corresponding monoesters (transesterification) must be carried out.
Since the biological sources, in particular fish oil, are not available without restrictions, it is of interest to isolate the PUFAs from microorganisms, such as bacteria, algae etc., which have stored these fatty acids within the cells or in the cell membranes as lipids.
There is great industrial interest in industrial methods for the preparative-scale production of fatty esters, in particular of nutritionally important fatty esters, preferably polyunsaturated fatty esters. Lipids contain fatty acids, mostly bound in glycerides (mono-, di- and triglycerides), phosphatides, glycolipids and aminolipids. These bound fatty acids and free native fatty acids and their derivatives differ firstly in their frequency of occurrence in biological sources, and secondly in their activity on the human organism.
Native fatty acids and their derivatives are produced from biological sources, in addition to by classical solvent extraction of the corresponding lipids, in particular by extraction using compressed gases (for example supercritical carbon dioxide, etc.). This method termed SFE (supercritical fluid extraction), ensuring biological compatibility, is a mild much-described extraction method which is used in routine analysis and process engineering.
Extracted lipids cannot be separated directly by chromatography into the individual triglycerides, since generally a permutation of numerous naturally occurring fatty acids at the three positions of the triglyceride leads to a multiplicity of compounds which can only be separated chromatographically with difficulty.
The fatty acids are therefore extracted by means of a preceding or following reaction via cleavage of the lipids (fat cleavage) into the individual fatty acids by means of (catalytic) transesterification to form the esters of lower alcohols or by means of (catalytic) hydrolysis to give the free acids, also in the presence of a compressed gas in the reaction medium (SFR=supercritical fluid reaction).
The substances which serve as catalysts here are organic acids (formic acid, acetic acid, citric acid, etc. xe2x80x9cCoupling chemical derivatisation reactions with supercritical fluid extractionxe2x80x9d, J. A. Fields, J. Chromatog. A, 785 (1997), pp. 239-249) and solid catalysts (e.g. Ion-exchange resins (C. Vieville, Z. Mouloungui, A. Gaset; Colloq.xe2x80x94Inst. Natl. Rech. Agron. (1995), 71 (Valorisations Non-Alimentraires des Grandes Productions Agricoles), 179-82; acidic aluminum oxide (B. W. Wenclawiak, M. Krappe, A. Otterbach; J. Chromatogr. A (1997), 785, 263-267)) or combinations thereof (C. Vieville, Z. Mouloungui, A. Gaset; Ind. Eng. Chem. Res. (1993), 32(9), 2065-8).
Enzymatic reactions using lipases are also known which, either in solution, or immobilized, perform the fat cleavage with subsequent extraction in the presence of supercritical gases (R. Hashizume, Y. Tanaka, H. Ooguchi, Y. Noguchi, T. Funada; JP-196722 and A. Marty, D. Combes, J. S. Condoret; Prog. Biotechnol. (1992), 8 (Biocatalysis in Non-conventional Media), 425-32).
In principle, the SFE and SFR methods can be carried out as continuous flow or batch methods. King et al. carry out extraction and transesterification in the presence of compressed gases as a batch method (J. W. King, J. E. France, J. M. Snyder; Fresenius J. Anal. Chem. (1992), 344, 474-478). In this case, firstly, extraction of lipids from a biological source takes place (here seed grains), with subsequent transesterification on the catalyst to form methyl esters. The aluminum oxide catalyst is physisorbed with ethanol. However, a disadvantage is the complex use of the samples on the catalyst, as a result of which reaction only takes place at the catalyst/sample interface. This is inadequate in the context of a preparative reaction.
King et al. describe a virtually quantitative conversion ( greater than 98%) of triglycerides to the methyl esters on immobilized lipase which is carried out as a continuous flow method. In this case corn oil and, as modifier, methanol are pumped to the carbon dioxide. This is also carried out for consistent biological sources such as soybean flakes (M. A. Jackson, J. W. King; J. Am. Oil. Chem. Soc. (1996), 73(3), 353-6).
However, the use of lipases in the continuous flow method has the disadvantage of conversion rates which are low with time.
All of the procedures described in the prior art have the disadvantage that they exhibit a spatial separation between extraction and reaction and thus do not comply with in situ preconditions and therefore do not achieve quantitative conversion. (Cf. also in the case of an esterification JP 61261398, JP 07062385 A2 and in the case of a hydrolysis K. Fujita, M. Himi; Nippon Kagaku Kaishi (1995), (1)). In addition, in the prior art, there is no advantageous inexpensive combination of reaction, extraction and chromatography.
The object of the present invention is to provide a method for the preparative production of unsaturated and saturated fatty esters and their selective isolation from biological sources.
The object is achieved by a method for the preparation and isolation of fatty esters from biological sources using continuous in-situ extraction, reaction chromatography. In the presence of a compressed gas stream and a 0.5 to 5% strength C1-C5 alcohol modifier
(a) the reaction takes place on an inert catalyst in complete contact with the biological source;
(b) the reaction products are chromatographed on the inert catalyst from (a) which has chromatographic retention and exclusively desorbs and elutes the reaction products;
(c) the desorbed and eluted reaction products from (b) are extracted.
The present method has advantages compared with known procedures:
The reaction products produced are safe with regards to health and food chemistry, since
1.) ethanol is preferably used as modifier and reaction partner and
2.) solid inert aluminum oxide is used as catalyst/stationary phase and
3.) carbon dioxide is used as reaction/extraction medium and mobile phase. Neither the starting materials nor the product thus come into contact with toxic substances at any instant of the method.
Carbon dioxide serves as protecting gas atmosphere to prevent oxidations and for mild extraction and elution.
In contrast to the mentioned methods, in the present case the toxicity of the substances used is so low that these may safely be used for preparing food additives or pharmaceutical products.
The reaction products occur in pure form as solid substance or in high concentration in a suitable solvent and can readily be further processed.
In addition, the reaction products in the inventive method are selectively separated off from the starting materials, the intermediates and the byproducts.
The method can be carried out continuously. In the case of liquid starting materials, they can be fed into the flow system.
Owing to the preferred use of the inexpensive aluminum oxide as inert catalyst/stationary phase, an economically expedient, industrial preparative scale can be carried out.
The biological source can be used directly. It is not necessary to limit the amount of biomass from the biological source and the method is therefore suitable for industrial use.
The method unites and combines the process steps extraction, reaction and chromatography to form a functional unit and can therefore decrease the costs of industrial use.
By carrying out the method as a continuous flow system the desired product is constantly removed from the reaction equilibrium and permits a theoretical yield of virtually 100%. This is impossible in a batch method or via classical organic transesterification.
xe2x80x9cIn-situ extraction, reaction and chromatographyxe2x80x9d for the purposes of this invention means that the fatty esters are provided, chromatographed and extracted from the biological source in situ using compressed gases (abbreviation: SF-REC).
For this purpose a specific catalyst is required which is triturated and/or mixed in situ with the biological source, and effects the cleavage of the lipids with reaction (transesterification) with an alcohol to form the fatty ester. In addition, the inventive catalyst acts as stationary phase in which the fatty esters produced are selectively desorbed from the catalyst and are eluted in the presence of the compressed gas. Therefore, the inventive catalyst must have a chromatographic retention at which the product is not adsorbed.
The inventive parameters (conditions) are chosen so that the lipids remain on the catalyst and the fatty esters are selectively eluted. These specific parameters are explained in the examples.
The inventive method of in-situ reaction, extraction chromatography is carried out as a continuous method. Use can also be made synonymously of the term xe2x80x9c(continuous) batch flow methodxe2x80x9d. For the purposes of this invention, the term continuous method is understood as a continuous flow system in which, with progressing reaction, in the gas/modifier stream, the reaction products are chromatographed and extracted on the solid or consistent biological source in contact with the inert catalyst. A liquid biological source can be added to the gas/ethanol stream and ensures a continuous method.
The term xe2x80x9ccompressed gasesxe2x80x9d for the purposes of this invention comprises liquid, supercritical and biphasic or subcritical gases or gas mixtures. Here, those gases are expressly incorporated which are known to those skilled in the art in the sector of SFE and SFR techniques. To this extent, the compressed gas serves for extraction and is used as reaction medium. For the purposes of this invention, the gas also serves as mobile phase and is used as extraction medium. Particular preference is given to compressed carbon dioxide.
For the purposes of this invention, xe2x80x9cmodifierxe2x80x9d means an additional stream in the presence of the compressed gas. In the context of this invention, 0.5-5% by volume of lower alcohols are used. Preference is given to an ethanol modifier of 0.5-5% by volume. Particular preference is given to 1% by volume of ethanol, preferably technical-grade ethanol. For the purposes of this invention, the modifier serves as reactant for transesterification of lipids on the inert catalyst.
For the purposes of this invention, fatty esters are obtainable from all branched and unbranched fatty acids and fatty acid derivatives, such as hydroxy fatty acids, which have a carbon chain of at least 12 carbon atoms. Preferred fatty esters are ethyl esters of fatty acids, since the alcohol required for their preparation has the lowest toxicity of the lower alcohols. The invention can be carried out for unbranched or branched C1-C5 alcohols.
Starting materials are preferably lipids from biological sources and other bound fatty acids; reaction products are fatty esters. Since, preferably, ethanol is used as reactant and modifier for the reaction (transesterification), ethyl esters of fatty acids are preferably obtained.
Obviously, free fatty acids and their salts in the biological sources are converted into their fatty esters.
In the context of this invention, complete transesterification means the conversion of all fatty acids present in the starting materials into the corresponding ethyl esters of fatty acids, with unreacted starting materials continuing to remain (adsorption) on the inert catalyst.
Inert catalyst, for the purposes of this invention, means that this catalyst in complete contact with the biological source firstly accelerates the reaction (transesterification) to the reaction products, and secondly serves as stationary phase. For this purpose the catalyst must have chromatographic retention. The catalyst may not have toxic activity on the biological source and therefore is present inert toward the reaction. Commercially conventional aluminum oxide has proved advantageous and inexpensive, which aluminum oxide in addition, can be readily mixed and/or triturated with the biological source, if appropriate together with other aids and additives (for example sea sand).
In principle, in the inventive method, any biological source can be employed and used. Obviously, biological sources are advantageous which are rich in native fatty acids as such or in the form of fatty esters, in particular lipids. If polyunsaturated fatty acids are desired, appropriate biological sources must be used. The term biological source is therefore preferably to be applied to microorganisms which can easily be cultured. In this case, preference is given to microorganisms having a high content of polyunsaturated fatty acids (PUFAs), which can readily be disintegrated. This may be chemically, enzymatically, but preferably mechanically. Particular preference is given to microorganisms having a fatty acid spectrum which predominantly comprises one or only a few bound or native fatty acids. Suitable organisms are preferably those hereinafter:
(The source used for the underlying systematics for group 1: Handbook of Protoctista, Margulis, Corliss, Melkonian, Chapman, Jones and Bartlett Publishers, Boston (1990), and for group 2: Industrial Applications of Single Cell Oils, Kyle and Ratledge, American Oil Chemist"" Society, Champaign, Ill., 1992).
1st group: Microalgae and protozoa (=protists)
Phylum: Ciliophora
Genus: Tetrahymena, Colpidium, Parauronema, Paramecium
Phylum: Labyrinthulomycota
Genus: Ulkenia, Thraustochytrium, Schizochytrium
Phylum: Dinoflagellata
Genus: Crypthecodinium, Gymnodinium, Gonyoaulax
Phylum: Euglenida
Genus: Euglena
Phylum: Bacillariophyta
Genus: Nitzschia, Navicula, Cyclotella
2nd group: Fungi
Genus: Mortierella, Cunninghamella, Mucor, Phytium
3rd group: Bacteria
Genus: Butyrivibrio, Lactobacillus